Saline-alkali tolerant monolith KM2-2 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a saline-alkaline tolerant monolith KM2-2, which is preserved in the China Center for Type Culture Collection on July 31, 2024, the preservation number of the saline-alkaline tolerant monolith KM2-2 is CCTCC NO: M 20241700, the preservation number of the saline-alkaline tolerant monolith KM2-2 is CCTCC NO: M 20241700, and the saline-alkaline tolerant monolith KM2-2 is preserved in the China Center for Type Culture Collection on July 31, 2024. The invention further provides an application of the monolith KM2-2. The monolith KM2-2 provided by the invention has good salt resistance and alkali resistance, can be grown and cultured in a saline-alkali environment, and can improve the saline-alkali environment, so that comprehensive utilization and comprehensive treatment of the saline-alkali environment are realized.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and for example, to a salt- and alkali-tolerant monostellate algae KM2-2 and applications thereof. Background Art

[0002] At present, salinization of land and water has become a global problem. Therefore, the comprehensive utilization and comprehensive management of saline-alkali environment is not only a necessary measure to promote economic development and ecological environment restoration, but also an important way to ensure national food security.

[0003] In recent years, the use of saline-alkali water to cultivate aquatic animals such as shrimp and tilapia has achieved improvements in economic benefits, social benefits, and ecological benefits. It also proves that saline-alkali water aquaculture is not only an important way to increase the supply of aquatic products and arable land resources, but also an important measure to implement the "big food concept", which is of great significance to expanding the space for fishery development and ensuring food security. However, there are no reports on the use of saline-alkali environment to cultivate microalgae.

[0004] Microalgae are tiny single-cell algae that can not only cooperate with other organisms in the soil to promote the improvement of soil structure, but also improve the pH of the soil and increase the permeability of the soil through secretions, thereby improving the water use efficiency of the land and promoting plant growth. In general, microalgae can take advantage of their own growth in the soil, improve the soil environment, promote plant growth, and lay a solid foundation for the overall improvement of the land. However, due to the high salt and alkalinity characteristics of saline-alkali environments, they are not conducive to the growth of microalgae, which limits the application of microalgae in the comprehensive utilization and comprehensive management of saline-alkali environments.

[0005] In summary, there is an urgent need for a microalgae with good salt and alkali resistance, which can be grown and cultivated in a saline-alkali environment, so as to achieve the comprehensive utilization and comprehensive management of the saline-alkali environment, and enable microalgae management technology to play a greater role in technical fields such as agriculture and environmental protection. Summary of the invention

[0006] The purpose of the present disclosure is to overcome the deficiencies in the prior art and to provide a salt- and alkali-tolerant monostar algae KM2-2 and its application. The monostar algae KM2-2 has good salt tolerance and alkali tolerance, can utilize the saline-alkali environment for growth and cultivation, and improve the saline-alkali environment, thereby realizing the comprehensive utilization and comprehensive management of the saline-alkali environment.

[0007] The above purpose is achieved through the following technical solutions:

[0008] On the one hand, a salt- and alkali-tolerant Coelastrella saipanensis KM2-2 is provided. The Coelastrella saipanensis KM2-2 was deposited in the China Center for Type Culture Collection on July 31, 2024, and its deposit number is CCTCC NO: M20241700.

[0009] In this regard, it is worth noting that the monostar algae KM2-2 provided by the present disclosure has good salt tolerance and alkali tolerance.

[0010] On the other hand, there is provided a use of the monostar algae KM2-2 as described in any one of the above embodiments in growth and cultivation in a saline-alkali environment.

[0011] In some embodiments, the saline environment includes at least one of saline soil and saline water.

[0012] In some examples, the salt concentration of the saline-alkali environment is 10-30 g / L.

[0013] In some examples, the pH of the saline environment is greater than 7 and less than or equal to 9.5.

[0014] In some embodiments, the growth and culture conditions include: a light-dark cycle of 8 to 16 h / 8 to 16 h, a light intensity of 30 to 70 μmol·m -2 ·s -1 , the culture temperature is 24-26°C, and the culture speed is 150-180rpm.

[0015] In some examples, the light-dark cycle is 12h / 12h.

[0016] In some embodiments, the growing culture comprises at least one of mixotrophic growth and autotrophic growth.

[0017] In this regard, it should be noted that, since the monostar algae KM2-2 provided by the present disclosure has good salt tolerance and alkali tolerance, it can be grown and cultured in a saline-alkali environment, thereby realizing the comprehensive utilization of the saline-alkali environment.

[0018] On the other hand, a use of the monostar algae KM2-2 as described in any one of the above embodiments in improving a saline-alkali environment is provided.

[0019] In some embodiments, the saline environment includes at least one of saline soil and saline water.

[0020] In some examples, the salt concentration of the saline-alkali environment is 10-30 g / L.

[0021] In some examples, the pH of the saline environment is greater than 7 and less than or equal to 9.5.

[0022] In this regard, it should be noted that microalgae can not only cooperate with other organisms in the soil to promote the improvement of soil structure, but also improve the pH of the soil and increase the permeability of the soil through secretions, thereby improving the water use efficiency of the land and promoting plant growth; on this basis, since the monostar algae KM2-2 provided by the present disclosure has good salt tolerance and alkali tolerance, it can grow and culture in a saline-alkali environment and improve the saline-alkali environment, thereby realizing the comprehensive management of the saline-alkali environment.

[0023] On the other hand, a use of the monostar algae KM2-2 as described in any one of the above embodiments in the development of genes for salt tolerance and / or alkali resistance is provided.

[0024] In some embodiments, when the monostar algae KM2-2 is used only for gene development for salt tolerance, the salt concentration of the salt tolerance is 10 to 50 g / L, for example, 10 to 30 g / L.

[0025] In other embodiments, when the monostar algae KM2-2 is used only for gene development for alkali resistance, the alkali resistance pH is greater than 7 and less than or equal to 9.5.

[0026] In some other embodiments, when the monostar algae KM2-2 is used in the development of genes for salt tolerance and alkali tolerance, the salt concentration of the salt tolerance is 10-30 g / L; the pH of the alkali tolerance is greater than 7 and less than or equal to 9.5.

[0027] On the other hand, a use of the monostar algae KM2-2 as described in any one of the above embodiments in the preparation of high-salt and / or high-alkali products is provided.

[0028] In some embodiments, the product comprises at least one of a feed and a food.

[0029] On the other hand, a use of the monostar algae KM2-2 as described in any one of the above embodiments in promoting plant growth under salt stress and / or alkaline stress is provided.

[0030] In this regard, it should be noted that microalgae can not only cooperate with other organisms in the soil to promote the improvement of soil structure, but also improve the pH of the soil and increase the permeability of the soil through secretions, thereby improving the water use efficiency of the land and promoting plant growth; on this basis, since the monostar algae KM2-2 provided by the present disclosure has good salt tolerance and alkali tolerance, it can promote plant growth under salt stress and / or alkali stress.

[0031] The beneficial effects of the present disclosure are:

[0032] 1. The monostar algae KM2-2 provided in the present invention has good salt tolerance and alkali tolerance.

[0033] 2. The monostar algae KM2-2 provided in the present disclosure can utilize the saline-alkali environment for growth and cultivation, and improve the saline-alkali environment, thereby realizing the comprehensive utilization and comprehensive management of the saline-alkali environment.

[0034] 3. The monostar algae KM2-2 provided in the present disclosure is capable of promoting plant growth under salt stress and / or alkaline stress.

[0035] Biological Deposit

[0036] The present invention provides a Coelastrella saipanensis KM2-2, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024, with a deposit number of CCTCC NO: M 20241700. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the postal code is 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the morphology of algal cells of the algae strain isolated in Part 1 of Example 3;

[0038] Figure 2 The transmission electron microscope ultrastructure image of the algae cells of the algae strain isolated in Part 1 of Example 3;

[0039] Figure 3 This is a BLAST sequence alignment result diagram of the 18S rRNA gene sequence of the algae strain isolated in Part 2 of Example 3 in the NCBI database;

[0040] Figure 4 This is a BLAST sequence alignment result diagram of the tufA gene sequence of the algae strain isolated in Part 2 of Example 3 in the NCBI database;

[0041] Figure 5 The neighbor-joining tree constructed based on the gene sequence of 18S rRNA isolated from the algae strain in Part 2 of Example 3;

[0042] Figure 6 The neighbor-joining tree constructed based on the gene sequence of tufA isolated from the algae strain in Part 2 of Example 3;

[0043] Figure 7 The phenotypic effect of different salinity and alkalinity treatments on the growth dynamics of KM2-2 in Example 4 is shown in the figure;

[0044] Figure 8 This is a graph showing the effect of different salinity and alkalinity treatments on the cell status of KM2-2 for 7 days in Example 4;

[0045] Fig. 9 This is a graph showing the effect of different salinity and alkalinity treatments on the cell optical density of KM2-2 for 7 days in Example 4;

[0046] Fig.10 This is a graph showing the effect of different salinity and alkalinity treatments on the maximum photon efficiency of KM2-2 for 7 days in Example 4; wherein, Fig.10 A shows the changes in the maximum quantum efficiency of chlorophyll fluorescence of KM2-2 cells; Fig.10 B shows the change of the maximum photon efficiency of KM2-2;

[0047] Fig.11 This is a graph showing the effect of different salinity and alkalinity treatments on the actual light quantum yield of KM2-2 for 7 days in Example 4; wherein, Fig.11 A shows the changes in the actual light quantum yield of chlorophyll fluorescence in KM2-2 cells; Fig.11 B shows the change of actual light quantum yield of KM2-2;

[0048] Fig.12 This is a graph showing the effect of different salinities and alkalinities on the pH and salinity of the cell culture fluid of KM2-2 after 7 days of treatment in Example 4; wherein, Fig.12 A shows the change in pH of the cell culture medium of KM2-2; Fig.12 B shows the change in salinity of the cell culture medium of KM2-2;

[0049] Fig.13 The phenotypic effect of saline-alkali water treatment from different sources on the growth dynamics of KM2-2 in Example 5;

[0050] Fig.14 This is a graph showing the effect of saline-alkali water from different sources on the cell state of KM2-2 for 7 days in Example 5; wherein, Fig.14 A shows the effect of saline water from different sources without adding modified BG11 medium on the cell state of KM2-2; Fig.14 B shows the effect of adding saline-alkali water from different sources to the modified BG11 medium on the cell state of KM2-2;

[0051] Fig.15 This is a graph showing the effect of saline-alkali water from different sources on the cell optical density of KM2-2 for 7 days in Example 5;

[0052] Fig.16This is a graph showing the effect of saline-alkali water from different sources on the chlorophyll fluorescence of KM2-2 for 7 days in Example 5;

[0053] Fig.17 This is a graph showing the effect of saline-alkali water from different sources on the maximum photon efficiency and actual photon yield of KM2-2 after 7 days of treatment in Example 5; wherein, Fig.17 A shows the change of the maximum photon efficiency of KM2-2; Fig.17 B shows the change of actual light quantum yield of KM2-2;

[0054] Fig.18 This is a graph showing the effect of saline-alkali water from different sources on the pH and salinity of the cell culture fluid of KM2-2 for 7 days in Example 5; wherein, Fig.18 A shows the change in pH of the cell culture medium of KM2-2; Fig.18 B shows the change in salinity of the cell culture medium of KM2-2. DETAILED DESCRIPTION

[0055] The technical solutions in some embodiments of the present disclosure are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0056] Unless the context requires otherwise, throughout the specification and claims, the term "comprising" is to be construed in an open, inclusive sense, ie, meaning "including, but not limited to."

[0057] When describing some embodiments, the expression "A and / or B" may be used. It is easy to understand that "A and / or B" includes the following three combinations: only A, only B, and a combination of A and B.

[0058] When describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B and C.

[0059] Example 1 Culture medium

[0060] 1. BG11 liquid culture medium, the formula of which is: sodium nitrate 1.4-1.6 g / L, K2HPO4·3H2O 0.03-0.05 g / L, MgSO4·7H2O 0.065-0.085 g / L, CaCl2·2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ammonium ferric citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, boric acid 0.0027-0.0029 g / L, MnCl2·H2O 0.0017-0.0019 g / L, ZnSO4·7H2O 0.00021~0.00023g / L, CuSO4·5H2O0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L and Co(NO3)2·6H2O 0.00004~0.00006g / L, the solvent is ddH2O; the pH value is 6.8~7.2.

[0061] 2.BG11 solid plate, its formula is: agar powder 12-17 g / L, sodium nitrate 1.4-1.6 g / L, K2HPO4·3H2O 0.03-0.05 g / L, MgSO4·7H2O 0.065-0.085 g / L, CaCl2·2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ammonium ferric citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, boric acid 0.0027-0.0029 g / L, MnCl2·H2O 0.0017~0.0019g / L, ZnSO4·7H2O0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O 0.0003~0.0005g / L and Co(NO3)2·6H2O0.00004~0.00006g / L; pH value is 6.8~7.2.

[0062] 3. Improved BG11 liquid culture medium, the formula of which is: glucose 10.0-30.0 g / L, sodium nitrate 1.4-1.6 g / L, K2HPO4·3H2O 0.03-0.05 g / L, MgSO4·7H2O 0.065-0.085 g / L, CaCl2·2H2O 0.026-0.046 g / L, citric acid 0.005-0.007 g / L, ammonium ferric citrate 0.005-0.007 g / L, EDTA 0.0005-0.0015 g / L, sodium carbonate 0.015-0.025 g / L, boric acid 0.0027-0.0029 g / L, MnCl2·H2O 0.0017~0.0019g / L, ZnSO4·7H2O0.00021~0.00023g / L, CuSO4·5H2O 0.00007~0.00009g / L, Na2MoO4·2H2O0.0003~0.0005g / L and Co(NO3)2·6H2O 0.00004~0.00006g / L, the solvent is ddH2O; the pH value is 6.8~7.2.

[0063] Example 2 Collection, activation, separation, cultivation and purification of algae

[0064] 1. Collection of algae species

[0065] On January 12, 2024, water samples were collected from Shilin Scenic Area, Shilin Yi Autonomous County, Kunming City, Yunnan Province (longitude: 103°32′76″, latitude: 24°81′61″).

[0066] 2. Activation of algae species

[0067] Transfer the collected water sample to a 15mL centrifuge tube and let it stand for 1 to 2 hours. Gently remove most of the supernatant, retain 2 to 3mL of liquid at the bottom, mix thoroughly, and culture on a shaker at 25±1℃ and 150 to 180rpm for 6 hours to fully activate the sample.

[0068] 3. Isolation and cultivation of algae

[0069] 10 μL of the activated sample was dropped onto a glass slide. After confirming that it contained suspected target algae cells under a microscope, a single cell was picked up under a microscope using the capillary siphon separation method. The sample was repeatedly aspirated, examined under a microscope, and diluted until only a single target algae cell was contained in the water droplet. The sample was then transferred to a 96-well plate containing 100 μL BG11 liquid culture medium (without antibiotics) and cultured statically. The light-dark cycle was 12 h / 12 ​​h and the light intensity was 20-30 μmol / m -2 / s -1The culture temperature was 25±1°C. During static culture, BG11 liquid culture medium was appropriately supplemented to about 100 μL according to the situation.

[0070] 4. Purification of Algae

[0071] After 20-30 days of static culture of a single target algae cell, the algae growth status was examined under a microscope. If the algae cells grew well, the cells were diluted to 10 0 , 10 -1 , 10 -2 and 10 -3 The concentrations were mixed thoroughly, and 200 μL of each gradient dilution was taken and spread on a plate containing 50 mg / L ampicillin (Amp 50+ )、50mg / L kanamycin (Kanamycin 50+ ) and 100mg / L cephalosporin (Cef 100 + ) on a BG11 solid plate, and place the plate in an inverted culture in a constant temperature light incubator with a light-dark cycle of 12 h / 12 ​​h and a light intensity of 10-30 μmol / m -2 / s -1 , the culture temperature is 25±1℃, inverted culture for 7-15 days until a single algae colony grows, and observe the growth of single algae colonies and bacteria on the plate. If there are obvious bacteria on the plate, pick a single algae colony and add it to 200μL BG11 liquid culture medium (containing Amp 100+ , Kan 50+ , Cef 100+ ) Mix by pipetting and dilute to 10 -1 and 10 -2 Mix thoroughly and re-apply to the BG11 solid plate (which also contains Amp 100+ , Kan 50+ , Cef 100+ ) were cultured on the plate. The algae were purified by plating the plates several times until there were no obvious foreign bacteria on the plates and single algae colonies. Then, single algae colonies were selected and placed on the modified BG11 liquid medium (containing Amp 25+ , Cef 50+ ), inoculated and cultured step by step at a ratio of 1:10, with a culture temperature of 25±1°C, a culture speed of 150-180 rpm, a light-dark cycle of 12h / 12h, and a light intensity of 30-50μmol / m -2 / s -1 , to reserve algal cells for subsequent steps.

[0072] Example 3 Identification and preservation of algae strains

[0073] 1. Morphological observation of algae strains

[0074] A small amount of purified fresh algae cells were taken and observed for morphology under a Leica DM5000 inverted microscope (Leica, Germany), and images were collected using a Leica DFC320 CCD, and differential interference contrast (DIC) and phase contrast (PH) photography were performed. At the same time, a small amount of fresh algae cells were pre-fixed with 3% glutaraldehyde, re-fixed with 1% osmium tetroxide, dehydrated in acetone step by step, and embedded in Epon812. Semi-thin sections were stained with toluidine blue for optical positioning, ultra-thin sections were made with a diamond knife, stained with uranyl acetate and lead citrate, and the cell ultrastructure was observed using a JEM-1400FLASH transmission electron microscope.

[0075] The morphology of the isolated microalgae was observed by optical microscopy and electron microscopy. Figures 1-2 shown.

[0076] The isolated algae strain is a single-cell green microalgae. The microalgae cells are spherical, ellipsoidal or spindle-shaped with blunt ends. The size of young cells is about (6-9) μm×(5-7) μm, the size of mature cells is about 8-14 μm, and the cell wall is relatively thick.

[0077] 2. Molecular Identification of Algal Strains

[0078] The purified algae were centrifuged at 5000 rpm for 5 min to collect the algae, resuspended with sterile water several times to remove the liquid culture medium, and the algae were collected for further molecular identification. The Ezup column-type plant tissue genomic DNA extraction kit of Sangon Biotech Co., Ltd. was used to extract the algae genome according to the instructions; the New England Biolabs High-Fidelity DNA Polymerase was used for PCR amplification.

[0079] The 50 μL PCR reaction system is: 25 ng of genomic DNA from KM2-2 algae strain, Reaction Buffer 10μL, 10mM dNTP 1μL, 10μM upstream and downstream primers 2.5μL each, High-Fidelity DNA Polymerase 0.5 μL, make up to 50 μL with double distilled water.

[0080] PCR molecular identification was performed using two genes, 18S ribosomal RNA gene (18S rRNA) and translation elongation factor Tu (tufA). The reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

[0081] Among them, there are two pairs of molecular identification primers, namely:

[0082] 1) Upstream primer 5'-AACCTGGTTGATCCTGCCAGT-3' and downstream primer 5'-TGATCCTTCTGCAGGTTCACCTAC-3' for amplifying the 18S rRNA region;

[0083] 2) The upstream primer 5'-TGAAACAGAAMAWCGTCATTATGC-3' and the downstream primer 5'-CCTTCNCGAATMGCRAAWCGC-3' were used to amplify the tufA region.

[0084] The products obtained by PCR amplification of the 18S rRNA region and tufA region were recovered and purified using a Takara gel recovery kit and sent to Chengdu Branch of Sangon Biotechnology (Shanghai) Co., Ltd. for sequencing. The sequencing results were submitted to the National Center for Biotechnology Information (NCBI) database for Nucleotide BLSAT sequence alignment. The results were as follows: Figure 3 and Figure 4 It can be found that the gene sequencing results of the product corresponding to the 18SrRNA region and the product corresponding to the tufA region have a query coverage (Query Cover) of 98% and 98% respectively, and a consistency (Per.Ident) of 99.77% and 96.08% respectively, and the E value (E value) is 0 with the corresponding sequence of Coelastrella saipanensis.

[0085] The gene sequences of 18S rRNA and tufA of the algae strain are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The joint neighbor-joining phylogenetic trees of the gene sequences of 18S rRNA and tufA of the algae strain are shown in Figure 5 and Figure 6As shown, the NCBI accession numbers of the 18S rRNA and tufA gene sequences of the algae strain are PQ108912.1 and PQ127014.1, respectively.

[0086] Analysis showed that this algae strain is in the same evolutionary branch as Coelastrella saipanensis.

[0087] 3. Preservation of algal strains

[0088] Based on the results of morphological observation and molecular identification, the algae strain obtained in this example is Coelastrella saipanensis, and the algae strain is named Coelastrella saipanensis KM2-2, which was deposited in the China Center for Type Culture Collection (CCTCC) on July 31, 2024. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072, and its deposit number is CCTCC NO: M 20241700.

[0089] Example 4 Salt-alkali tolerance test of monostar algae KM2-2

[0090] 1. Experimental Materials

[0091] The monostar algae KM2-2 (ie, KM2-2) in Example 3 was used in this example.

[0092] 2. Experimental Methods

[0093] 2.1 Treatment with different salinity and alkalinity

[0094] KM2-2 was cultured according to the culture conditions of Part 4 of Example 2 to the late logarithmic growth phase, and fresh modified BG11 liquid culture medium was replaced, and the initial concentration of algal cells was adjusted to OD680 = 0.5, and then distributed into 50 mL Erlenmeyer flasks for subsequent treatment experiments.

[0095] Different alkalinity treatments were performed with pH=7.0 and pH=9.5; different salinity treatments were performed with NaCl at concentrations of 0 g / L, 10 g / L, 30 g / L and 50 g / L, and correspondingly, the salinities were 0, 1%, 3% and 5%, respectively; and different alkalinity treatments and different salinities were cross-combined; the culture temperature was 25±1°C, the culture speed was 150-180 rpm, and the light intensity was 30-70 μmol·m -2 ·s -1 , the light-dark cycle was 12h / 12h, and the culture was continued for 7 days.

[0096] 2.2 Determination of microalgae growth curve

[0097] A certain amount of fresh algae liquid was taken, and the optical density of KM2-2 cells at 680 nm was measured using a microplate reader (Metash, UV-9000, China), and a growth curve was drawn.

[0098] 2.3 Chlorophyll fluorescence analysis

[0099] Chlorophyll fluorescence was measured using MAXI-IMAGING-PAM (WALZ, Germany). After mixing the samples, 200 μL was pipetted into a black 96-well plate and the chlorophyll fluorescence parameters were measured after dark adaptation for 5 min.

[0100] 2.4 Determination of pH and salinity

[0101] The pH was measured using a pH meter, and the salinity was measured using a salinity meter.

[0102] 3. Experimental results

[0103] This example studies the effects of different alkalinity (initial pH of 7.0 and 9.5, respectively) and different salinity (NaCl concentrations of 0 g / L, 10 g / L, 30 g / L and 50 g / L, respectively, and correspondingly, salinity of 0, 1%, 3% and 5%) on the growth state of KM2-2 algae cells after continuous culture for 7 days. The results are shown in FIG. Figures 7 to 12 shown.

[0104] 3.1 Test of tolerance of KM2-2 to salt stress

[0105] When the initial pH of the culture solution is 7.0, the specific performance is as follows:

[0106] As the culture time increases, under the treatment of 1% NaCl, the color of the algae culture solution gradually changes from light green to dark green (e.g. Figure 7 After 7 days of continuous culture, the number of cells increased significantly under the 1% NaCl treatment compared with the 0% NaCl treatment (as shown in Figure 8 The cell optical density was maintained at 0.8 to 0.9 (as shown in Fig. 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are slightly reduced (as shown Figures 10-11 This indicates that when the initial pH of the culture solution is 7.0, the NaCl treatment with a salinity of 1% promotes the growth of KM2-2.

[0107] As the culture time increases, under the treatment of 3% NaCl, the color of the algae culture solution changes from light green to dark green on days 0 to 5, and from dark green to light green on days 5 to 7 (e.g. Figure 7 After 7 days of continuous culture, the number of cells increased slightly compared with the NaCl treatment with a salinity of 0 (as shown in Figure 8 The cell optical density is maintained at 0.5 to 0.6 (as shown in Fig. 9 As shown), the maximum photon efficiency Fv / Fm and the actual photon yield Y(II) are reduced (as shown Figures 10-11 ). This indicates that when the initial pH of the culture solution is 7.0, the 3% NaCl treatment promotes the growth of KM2-2 in 0 to 5 days, but cannot continue to promote its growth if the culture time is too long.

[0108] As the culture time increases, under the treatment of 5% NaCl, the color of the algae culture solution changes from light green to brown green (e.g. Figure 7 After 7 days of continuous culture, the number of cells decreased significantly compared with the NaCl treatment with a salinity of 0 (as shown in Figure 8 The cell optical density is maintained at 0.4 to 0.5 (as shown in Fig. 9 As shown), the maximum photon efficiency Fv / Fm and the actual photon yield Y(II) are significantly reduced (as shown Figures 10-11 ). This indicates that when the initial pH of the culture medium is 7.0, the treatment with 5% NaCl can only maintain the survival of KM2-2, but cannot promote its cell growth.

[0109] When the initial pH of the culture medium was 7.0, the pH changed to 9.53, 9.32, 8.93, and 8.70 respectively after 7 days of continuous culture under the treatment of 0%, 1%, 3%, and 5% NaCl salinity (e.g. Fig.12 A), its salinity is basically maintained at the initial addition concentration (as shown in Fig.12 B).

[0110] In summary, compared with the NaCl treatment with a salinity of 1% to 5%, the growth rate of KM2-2 was the fastest and the growth state was the best under the NaCl treatment with a salinity of 0; in addition, when the initial pH of the culture medium was 7.0, KM2-2 had good tolerance to NaCl with a salinity of 1% to 3%, and had a certain tolerance to NaCl with a salinity of 5%.

[0111] 3.2 Test of tolerance of KM2-2 to heavy alkaline water stress

[0112] When the initial pH of the culture solution is 9.5, the specific performance is as follows:

[0113] As the culture time increases, under the treatment of 1% NaCl, the color of the algae culture solution gradually changes from light green to dark green (e.g. Figure 7 After 7 days of continuous culture, the number of cells increased significantly compared with the NaCl treatment with a salinity of 0 (as shown in Figure 8 The cell optical density was maintained at 0.8 to 0.9 (as shown in Fig. 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are slightly reduced (as shown Figures 10-11 This indicates that when the initial pH of the culture solution is 9.5, the NaCl treatment with a salinity of 1% promotes the growth of KM2-2.

[0114] As the culture time increases, under the treatment of 3% NaCl, the color of the algae culture solution changes from light green to brown green (e.g. Figure 7 After 7 days of continuous culture, the number of cells decreased significantly compared with the NaCl treatment with a salinity of 0 (as shown in Figure 8 The cell optical density is maintained at 0.4 to 0.5 (as shown in Fig. 9 As shown), the maximum photon efficiency Fv / Fm and the actual photon yield Y(II) are significantly reduced (as shown Figures 10-11 ). This indicates that when the initial pH of the culture medium is 9.5, the NaCl treatment with a salinity of 3% can only maintain the survival of KM2-2, but cannot promote its cell growth.

[0115] As the culture time increases, under the treatment of 5% NaCl, the color of the algae culture solution changes from light green to brownish yellow (e.g. Figure 7 After 7 days of continuous culture, the number of cells decreased significantly compared with the NaCl treatment with a salinity of 0 (as shown in Figure 8 The cell optical density is maintained at 0.3-0.4 (as shown in Fig. 9 As shown), the maximum quantum efficiency Fv / Fm and the actual quantum yield Y(II) are almost undetectable (as shown Figures 10-11 ). This indicates that when the initial pH of the culture medium is 9.5, the treatment with 5% NaCl inhibits the growth of KM2-2 and the cells gradually die.

[0116] When the initial pH of the culture medium was 9.5, the pH changed to 9.49, 9.33, 8.83, and 7.85 respectively after 7 days of continuous culture under the treatment of 0%, 1%, 3%, and 5% NaCl salinity (e.g. Fig.12 A), its salinity is basically maintained at the initial addition concentration (as shown in Fig.12 B).

[0117] In summary, compared with the NaCl treatment with a salinity of 1% to 5%, the growth rate of KM2-2 was the fastest and the growth state was the best under the NaCl treatment with a salinity of 0. This shows that when the initial pH of the culture medium is 9.5, the NaCl treatment with a salinity of 0 has no effect on the growth of KM2-2 at all; in addition, when the initial pH of the culture medium is 9.5, KM2-2 has good tolerance to NaCl with a salinity of 1%, has a certain tolerance to NaCl with a salinity of 3%, and has poor tolerance to NaCl with a salinity of 5%.

[0118] Example 5 Study on the effect of growth and cultivation of monostar algae KM2-2 using saline-alkali water

[0119] 1. Experimental Materials

[0120] The monostar algae KM2-2 (ie, KM2-2) in Example 3 was used in this example.

[0121] 2. Experimental Methods

[0122] 2.1 Treatment of saline-alkali water from different sources

[0123] Samples A, B, and C used for saline-alkali water treatment were collected in Atushi City, Kizilsu Kirgiz Autonomous Prefecture, Xinjiang Uygur Autonomous Region, among which samples A, B, and C were collected in Kumusak Village, Azak Town (longitude: 76°15′ east, latitude: 39°40′ north), Nierkul Salt Lake, Ang’ezi Village, Halajun Township (longitude: 75°31′ east, latitude: 39°39′ north), and Langan Village, Azak Town (longitude: 76°3′ east, latitude: 39°40′ north).

[0124] Treatment 1: KM2-2 was cultured to the late logarithmic growth phase according to the culture conditions of Part 4 of Example 2, and then transferred to samples A, B, and C for treatment, respectively, to obtain treatment 1-1, treatment 1-2, and treatment 1-3; ddH2O was used as control 1; the initial concentration of algal cells in each group was adjusted to OD680 = 0.5, and then divided into 50 mL triangular flasks for subsequent treatment experiments. The specific information of the culture solution of each group before treatment is shown in Table 1.

[0125] Table 1 Statistical information of each group of culture fluid in treatment 1 before treatment

[0126] Comparison 1 Process 1-1 Process 1-2 Process 1-3 Sample source <![CDATA[ddH2O]]> Sample A Sample B Sample C pH 7.00 8.50 8.00 8.55 salinity(%) 0 2.0 27.3 2.1

[0127] Treatment 2: Add modified BG11 medium to samples A, B, and C, sterilize with high pressure steam at 121°C for 15 min for later use; culture KM2-2 to the late logarithmic growth phase according to the culture conditions of Part 4 of Example 2, and transfer to sample A + modified BG11 medium, sample B + modified BG11 medium, and sample C + modified BG11 medium, respectively, to obtain treatment 2-1, treatment 2-2, and treatment 2-3; at the same time, a modified BG11 medium using ddH2O as a solvent was used as control 2; each group adjusted the initial concentration of algae cells to OD680 = 0.5, and then dispensed into 50 mL triangular flasks for subsequent treatment experiments. The specific information of each group of culture solution before treatment is shown in Table 2.

[0128] Table 2 Statistical information of each group of culture fluid in treatment 2 before treatment

[0129]

[0130]

[0131] It should be noted that the "improved BG11 culture medium" mentioned above is different from the "improved BG11 liquid culture medium" in the embodiments of the present disclosure only in that it does not contain the solvent ddH2O. It can be seen that the "adding improved BG11 culture medium to samples A, B, and C respectively" mentioned above can also be understood as "replacing the solvent ddH2O in the improved BG11 liquid culture medium with samples A, B, and C respectively"; similarly, the "improved BG11 culture medium using ddH2O as solvent" mentioned above is the "improved BG11 liquid culture medium" in the embodiments of the present disclosure.

[0132] During treatment 1 and treatment 2, the culture temperature of KM2-2 was 25 ± 1 °C, the culture speed was 150-180 rpm, and the light intensity was 30-70 μmol·m -2 ·s -1 , the light-dark cycle was 12h / 12h, and the culture was continued for 7 days.

[0133] The other experimental methods are the same as those in Example 4 and will not be described again here.

[0134] 3. Experimental results

[0135] 3.1 Study on the effect of autotrophic growth of KM2-2 using saline-alkali water from different sources

[0136] As shown in Table 1, the pH of the culture medium of treatment 1-1, treatment 1-2, and treatment 1-3 before treatment were 8.50, 8.00, and 8.55, respectively, and the salinity were 2.0%, 27.3%, and 2.1%, respectively; the pH of the culture medium of control 1 before treatment was 7.00, and the salinity was 0.

[0137] This example compares the effects of natural saline water samples A, B, and C from different sources on the autotrophic growth state of KM2-2 algae cells. Figures 13 to 18 When saline water is used directly as the culture medium, the specific performance is as follows:

[0138] As the culture time increases, the color of the algae culture solution in treatments 1-1 and 1-3 remains dark green from day 0 to day 7 (e.g. Fig.13 After 7 days of continuous culture, the number of cells in treatments 1-1 and 1-3 increased slightly compared to control 1 (as shown in Fig.14 A), the cell optical density is maintained at 0.5-0.6 (as shown in Fig.15 There were no significant differences in chlorophyll fluorescence parameters (as shown in Fig.16 This indicates that the saline-alkali water of sample A and sample C can maintain the activity of KM2-2 cells.

[0139] As the culture time increases, the color of the algae culture solution in treatment 1-2 changes from dark green to brown-green (e.g. Fig.13 After 7 days of continuous culture, the number of cells in treatment 1-2 was slightly reduced compared with control 1, treatment 1-1 and treatment 1-3 (as shown in Fig.14 A), the cell optical density value decreased slightly (as shown in Fig.15 As shown), chlorophyll fluorescence imaging, maximum light quantum efficiency Fv / Fm and actual light quantum yield Y(II) are almost undetectable (as shown Figures 16-17 As shown in Figure 2 ). This indicates that the salinity of sample B (27.3%) hinders the normal photosynthesis of KM2-2 and affects the normal growth and reproduction of its cells.

[0140] After 7 days of continuous culture, the pH and salinity of the cell culture medium of KM2-2 in treatments 1-1, 1-2, and 1-3 remained basically unchanged (e.g. Fig.18 shown).

[0141] In summary, KM2-2 has good tolerance to saline-alkali water samples A and C, and can be used for normal autotrophic growth, but its tolerance to saline-alkali water sample B is poor.

[0142] 3.2 Study on the application of KM2-2 in mixotrophic growth in saline-alkali water from different sources

[0143] After high temperature and high pressure sterilization, the culture fluids of treatment 2-1, treatment 2-2 and treatment 2-3 changed to caramel color, the pH decreased and the salinity increased. As shown in Table 2, the pH of the culture fluids of treatment 2-1, treatment 2-2 and treatment 2-3 before treatment were 6.42, 5.32 and 6.91 respectively, and the salinity was 3.1%, more than 28% and 3.2% respectively; the pH of the culture fluid of control 2 before treatment was 6.54 and the salinity was 1.1.

[0144] This example compares the effects of natural saline water samples A, B, and C from different sources on the mixotrophic growth state of KM2-2 algae cells. Figures 13 to 18 When saline water + improved BG11 medium is used as the culture medium, the specific performance is as follows:

[0145] As the culture time increases, the color of the algae culture solution in treatments 2-1 and 2-3 changes from green to dark green (e.g. Fig.13 After 7 days of continuous culture, the number of cells in treatment 2-1 and treatment 2-3 was comparable to that in control 2 (as shown in Fig.14 B), the cell optical density values ​​were 2.16 and 1.22 (as shown in Fig.15 There were no significant differences in chlorophyll fluorescence parameters (as shown in Fig.16 This indicates that the saline water of sample A and sample C can promote the growth of KM2-2 cells after adding the improved BG11 medium.

[0146] As the culture time increases, the color of the algae culture solution in treatment 2-2 changes from dark green to dark brown (e.g. Fig.13 After 7 days of continuous culture, the number of cells in treatment 2-2 decreased compared with control 2, treatment 2-1 and treatment 2-3 (as shown in Fig.14 B), the cell optical density value decreased (as shown in Fig.15 As shown), chlorophyll fluorescence imaging, maximum light quantum efficiency Fv / Fm and actual light quantum yield Y(II) are almost undetectable (as shown Figures 16-17 This indicates that even with the addition of the modified BG11 medium, the salinity of sample B of 27.3% hinders the normal photosynthesis of KM2-2 and affects the normal growth and reproduction of its cells.

[0147] After 7 days of continuous culture, the pH and salinity of the cell culture medium of KM2-2 in treatments 2-1, 2-2, and 2-3 remained basically unchanged (e.g. Fig.18 shown).

[0148] In summary, the addition of modified BG11 medium can enhance the tolerance of KM2-2 to natural saline-alkali water and promote the mixotrophic growth of its cells.

Claims

1. A salt-alkali tolerant Coelastrella saipanensis KM2-2, characterized in that: The monostar algae KM2-2 was deposited in the China Center for Type Culture Collection on July 31, 2024, and its preservation number is CCTCC NO: M20241700.

2. An application of the monostar algae KM2-2 as claimed in claim 1 in growth and cultivation in a saline-alkali environment.

3. The use according to claim 2, characterized in that: The saline-alkali environment includes at least one of saline-alkali soil and saline-alkali water.

4. A use of the monostar algae KM2-2 as claimed in claim 1 in improving saline-alkali environment.

5. The use according to claim 4, characterized in that: The saline-alkali environment includes at least one of saline-alkali soil and saline-alkali water.

6. Use of the monostar algae KM2-2 as claimed in claim 1 in the development of genes for salt tolerance and / or alkali tolerance.

7. Use of the monostar algae KM2-2 as claimed in claim 1 in the preparation of high-salt and / or high-alkali products.

8. The use according to claim 7, characterized in that: The product comprises at least one of feed and food.

9. Use of the monostar algae KM2-2 as claimed in claim 1 in promoting plant growth under salt stress and / or alkali stress.

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